Polymerase Chain Reaction- Basic

Principle

Promila SheoranPh.D. BiotechnologyGJU S&T Hisar

Principle of the PCRThe purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene.

The cycling reactions :•There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.1. Denaturation at 94°C : During the denaturation, the double strand melts open to single stranded DNA,

all enzymatic reactions stop (for example : the extension from a previous cycle).2. Annealing at 54°C :

The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.

3. Extension at 72°C :

This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

Inverse PCR

•Inverse PCR (IPCR) was designed for amplifying anonymous flanking genomic DNA regions.

• The technique involves the digestion of source DNA, circulation of restriction fragments, and amplification using oligonucleotides that prime the DNA synthesis directed away from the core region of a known sequence, i.e., opposite of the direction of primers used in normal or standard PCR.

•Prior to the invention of the polymerase chain reaction (PCR), the acquisition of a specific DNA fragment usually entailed the construction and screening of DNA libraries, and the traditional “walking” into flanking DNA fragments involved the successive probing of libraries with clones obtained in the prior screening.

•These time-consuming procedures could be replaced by IPCR. Because IPCR can be used to efficiently and rapidly amplify regions of unknown sequence flanking any identified segment of cDNA or genomic DNA, researchers do not need to construct and screen DNA libraries to obtain additional unidentified DNA sequence information using this technique.

• Some recombinant phage or plasmids may be unstable in bacteria and amplified libraries tend to lose them. IPCR eliminates this problem.

Anchored PCR

•When sequence of only one end of the desired segment or gene is known, the primer complementary to the 3’ end of this strand is used to produce several copies of only one strand of the desired segment.

•Now a poly–G is added to the 3’end (the end for which primer is not available) of the single-strand DNA copies produced by PCR.

•This allows the use of complementary homopolymer, poly-C, to be used as primer for copying the DNA single-strands generated by PCR, and give rise to the complete DNA duplex that can be amplified normally.

•This approach has been termed as anchored PCR and the poly-G tail added to the 3’ ends of the single strand copies is called anchor.

Asymmetric PCR

•Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other.

•It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal.

• PCR is carried out as usual, but with a great excess of the primers for the chosen strand.

•Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required

•To achieve this, quantities of the two primers for the 3’ borders of the target DNA segment are so adjusted in the reaction mixture that one of them is exhausted about 10 cycles or so cycles before the PCR is terminated.

•As a result, in the terminal 10 or so cycles only a single strand of DNA segment is copied; these single-strand copies are the starting materials for DNA sequencing.

•This variation of PCR is called asymmetric PCR.

RT-PCR

•This variation can be used to amplify RNA sequences into DNA duplexes. At first, a cDNA copy of the RNA is produced using the enzyme reverse transcriptase; this cDNA is then used for amplification.

•In case of eukaryotic mRNAs, an oligo-T sequence can serve as one of the primers for the PCR as well as for the reverse transcription step.

•The mRNA is copied into a single cDNA strand using an oligo-T as primer, which pairs with the 3’ poly- A tail of the mRNA.

•An oligo –G tail is now added to the 3’-end of the cDNA single-strand (the sequence of this end of cDNA is not known) by the enzyme terminal deoxynucleotidyl transferase.•The cDNA single-strand now has an oligo-T sequence at one end (5’ end) and an oligo-G sequence at the other (3’ end).

•We now use an oligo-C as a primer to copy the single-strand cDNA into cDNA duplex, which is amplified using an oligo-T and an oligo-C as primers.

PCR for site directed mutagenesis

•This technique is used for introducing mutations at the desired place in a DNA sequence by altering the sequences of primers.

•Since mutations are introduced only through primers, mutations are limited to the ends of the gene sequence.

•A variation of this technique allows mutations to any place of interest in the gene-the method is described as overlap extension PCR.

(i) In two separate PCR reactions, a particular gene is amplified into two separate segments. In both reactions there is one primer at the end of the gene and the other internal to the sequence.

(ii) The internal primers in two reactions are complementary to one another, so that the amplified products will have their ends internal to the original sequence.

(iii) These internal ends of products in two reactions will overlap. The sequences of internal primers can be suitably modified to introduce alterations in the overlap region.

(iv) The two PCR products are denatured and annealed, so that the internal ends in the overlap region will work as primers for each other.

Extension of these primers results in the formation of a complex gene, with the mutation incorporated into it

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